*4.5. Genetics*

The genetic makeup of a man with NOA may provide insight into his chances of successful sperm retrieval and can aid greatly in preoperative counseling. As per guidelines from the American Society of Reproductive Medicine and American Urologic Association, men with NOA or severe oligozoospermia (<5 million sperm/mL) should undergo karyotype analysis and screening for Y chromosome microdeletions [22,24]. It is well-known that the presence and location of a Y chromosome microdeletion in a man with NOA is helpful in predicting the chance of sperm retrieval [38]. Men with complete AZFa and AFZb deletions have sperm retrieval rates of zero, whereas men with AZFc deletions have reported sperm retrieval rates of up to 70% [38,39]. Furthermore, detection of Klinefelter Syndrome (47,XXY) provides favorable prognostic information, as these men tend to have similar or better rates of sperm retrieval as compared to other NOA men (ranging from 65–70% retrieval rates at our center over time) [9,37]. Moving forward, improved diagnostics such as whole exome sequencing may identify specific genetic abnormalities that may provide further prognostic information related to sperm retrieval success [40,41].

#### *4.6. History of Cryptorchidism/Orchiopexy*

Most men with a history of cryptorchidism have sperm in their ejaculate and nearly normal fertility (unilateral cryptorchidism) or mildly impaired fertility (bilateral cryptorchidism). Therefore, men who are azoospermic with a history of cryptorchidism are relatively unique. Our experience has demonstrated that men with azoospermia and a history of cryptorchidism/orchiopexy have unique anatomic features. These men often have a testis that is in a different anatomic configuration with the epididymis anterior, and the caput epididymis that may be inferior within the scrotum. Taken together with the lack of tunica vaginalis that is typically present after orchiopexy, exploration of the scrotum in these men can be difficult. Care must be taken to identify each anatomic structure, with special care taken to identify the testicular blood supply. The surface of the testis in these patients often has very prominent vessels that course in a longitudinal fashion, almost

suggesting a pattern of neovascularity. In some cases, the primary testicular blood supply does not enter in the standard location just medial to the caput epididymis.

Since most pexed testes have good sperm production, the azoospermic man with cryptorchidism/orchiopexy has a very different spermatogenic picture. Given the intraoperative observations we have made, it is possible that some of these men are azoospermic because of an alteration in testicular blood supply that occurred during, or as a consequence of, surgical orchiopexy.

Only limited reports of sperm retrieval success after orchiopexy for cryptorchidism have been published to-date, as of 2020 [42–46]. In these studies, combinations of testicular volume, unilateral and bilateral cryptorchidism have been debated as potential factors affecting the chance of sperm retrieval. Overall, a history of cryptorchidism has been suggested to be a favorable factor for sperm retrieval, with retrieval rates of 55–74% reported in these studies. We have typically found that most of the testis will be replaced with sclerotic tubules, with small distinct foci of sperm production, often in areas with Leydig cell hyperplasia (notable by the yellow color around the enlarged seminiferous tubules in these sites.)

#### *4.7. Other Factors*

Various studies have attempted to explore other factors including inhibin B levels, various gene products or transcripts in the ejaculate as well as anti-Müllerian hormone levels as predictors of successful sperm retrieval, but none have shown adequate association to warrant clinical application as predictors of sperm retrieval [37,47,48].

#### **5. Outcomes of cTESE vs. mTESE**

Numerous studies have endeavored to compare cTESE to mTESE. One of the first reviews summarized seven studies between 1999 and 2013 and reported sperm retrieval rates of 16.7–45.0% in the cTESE group and 42.9–63.0% in the mTESE group [49]. Shortly after, a meta-analysis of 15 studies with almost 2000 patients demonstrated that mTESE had a 1.5× greater likelihood of successful sperm retrieval compared to cTESE [47]. The strength of this meta-analysis was that it included only comparative studies, i.e., publications where the same selection of patients and same surgical/laboratory expertise was applied to compare patient outcomes in these settings. These data remain the most robust comparisons of testicular fine needle aspiration (TESA) with cTESE, showing a two-fold improvement in sperm retrieval rates with cTESE vs. TESA, as well as a higher rate of sperm retrieval with mTESE vs. cTESE (1.5× higher).

The review by Corona et al. published in 2019, included over 21,000 patients [28]. Since the authors included data from different patient cohorts with varying underlying etiologies for NOA, it is not possible to rely on the results tabulated as being a valid comparison of different sperm retrieval techniques. They did note a randomized trial which reported a retrieval rate of 42% (29/69 testicles) in the cTESE group versus 52% (36/69 testicles) in the mTESE group who had sperm retrieved at the time of surgery [48]. The authors sugges<sup>t</sup> that this difference was due to an ability to view larger tubules, obtain tubules from more vascularized areas, and the ability to map the testicle during mTESE. The findings of this one prospective RCT (randomized control trial) should be considered strongly as the randomized study design reduces confounding and affords the greatest exchangeability between study groups. The quality of a meta-analysis is only as good as the studies for which it summarizes, and in this case consisted almost entirely of observational data with many possible sources of bias.

#### **6. Optimizing Success**

Since NOA men may have focal areas of sperm production within their testis, preoperative optimization is a key strategy for successful sperm retrieval during TESE. As spermatogenesis takes approximately 74 days, it is important to ensure that patients do not have surgical intervention to the testicle including biopsy for at least 6 months prior to their

procedure [9]. Men with varicocele warrant consideration of possible varicocele repair, and may be considered in certain selected patients, especially couples with a younger female partner and lower FSH (less risk of Sertoli cell only syndrome) as well as those with previously documented sperm in the ejaculate or those with ample time to benefit from a return of sperm in their ejaculate. The caveat to varicocele repair is that it may take >6 months for return of sperm to the ejaculate, and only a small subset (<10%) may have adequate numbers of sperm to negate the need for surgical retrieval [9]. Again, of concern in these studies is the lack of a control group to compare to the patients who had varicocele repair for NOA. Some men with previously documented azoospermia will have rare sperm detected in a repeat semen analysis, especially if the concentrated pellet is more carefully examined ("extended sperm search") [50].

Hormonally, men with NOA typically have elevated gonadotropins (i.e., FSH), low serum testosterone (and subsequently low intratesticular testosterone), and mildly elevated estradiol levels [51]. This hormone profile lends itself to possible manipulation in an effort to increase intratesticular testosterone levels and spermatogenesis. Those with low serum testosterone and elevated estradiol (abnormal ratio of testosterone to estradiol) may have increased levels of aromatase, and therefore off-label use of aromatase inhibitors may increase serum testosterone levels, decrease estradiol, support spermatogenesis and increase intratesticular testosterone [52]. Other strategies for hormone optimization in NOA men include the use of selective estrogen receptor modulators (SERMs) and human chorionic gonadotrophic (hCG). Clomiphene citrate is a SERM that promotes gonadotropin release secondary to competitive binding of the estrogen receptor resulting in increased androgen production, and hCG works to directly stimulate luteinizing hormone (LH) receptors on Leydig cells in the testicle for androgen production [4]. Although normalizing serum testosterone levels (and thereby enhancing intratesticular sperm production) makes sense for men with low testosterone, the proof that such medical intervention helps sperm retrieval rates is limited. Most studies have been non-comparator trials, with the same limitations as noted for varicocele repair prior to TESE. Indeed, our non-randomized results showed that men with low T who were treated had a sperm retrieval rate of 51%, but the men with low T who were not treated had sperm found in 61% of cases (*p* = 0.3) [53].

Sperm processing, as previously described, is also a key aspect for optimization and identification of sperm. Using sharp scissors to finely mince the harvested tissue, followed by repeated aspiration of the tissue homogenate through a 24-gauge angiocath ensures that surgically retrieved tissue is adequately disrupted, and results in up to a 300-fold increase in detectable sperm within minutes of analysis in the operating room [9].

#### **7. The Future of Sperm Retrieval in NOA Men**

mTESE has been a revolutionary procedure for the treatment of men with NOA. However, there are relatively limited preoperative factors at this time that help to predict sperm retrieval. Therefore, there is ongoing reliance on dedicated surgical examination of testicular tissue in these patients as well as surgeon experience, preoperative optimization (hormone levels and varicocele repair), and teamwork with reliable and experienced laboratory personnel to increase success. Unfortunately, further processing of tissue in the laboratory rarely identifies sperm that was not detected on preliminary examination of a well-digested testicular tissue specimen in the operating room. Moving forward, new technologies for assisted reproduction, including microfluidics, cell sorting, or other micro-identification techniques could permit identification of rare sperm and possibly selection of optimal sperm from a limited pool to improve the likelihood of pregnancy success and live birth rate. Although testicular sperm have 0% normal morphology and are often qualitatively seen to be grossly abnormal, even immotile, from men with NOA, the 43% clinical pregnancy rate obtained with such sperm remains a remarkable reflection of the ability of sperm to contribute to a normal pregnancy with ICSI. Little further improvement in sperm retrieval rates is likely to occur with current surgical techniques, but the learning curve for finding rare sperm is difficult to master, and the care taken to

maintain testicular function is critical. Advanced imaging techniques to identify sites of sperm production preoperatively could greatly enhance the application of surgical sperm retrieval. Furthermore, a better understanding of the likely genetic components of NOA may allow non-surgical interventions to enhance sperm production, especially for men with maturation arrest as the presentation of NOA.

**Author Contributions:** Conceptualization: P.N.S.; writing: N.P., C.K. and P.N.S.; writing—review and editing: N.P., C.K. and P.N.S.; supervision P.N.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** N.P. and C.K. are supported in part by the Frederick J. and Theresa Dow Wallace Fund of the New York Community Trust.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Vanessa Dudley for her excellent illustrations.

**Conflicts of Interest:** The authors have no conflicts of interest to declare.
